Molecular detection of M. tuberculosis and M. avium complex: Real-time PCR assays are directly performed on clinical specimens to detect M. tuberculosis complex (MTBC) and Mycobacterium avium complex (MAC) DNA and to identify the different members of the MTBC within 24 (MTBC/MAC detection) to 48 hours (MTBC species identification) of specimen receipt.

Molecular detection of antimicrobial resistance: Pyrosequence analysis of a portion of the katG, inhA, and rpoB genes is performed on all primary patient specimens positive for MTBC. This assay allows for rapid identification of known mutations associated with resistance to rifampin (rpoB) and isoniazid (inhA, katG). Pyrosequence analysis of a portion of gyrA and gyrB genes can also be performed on primary specimens that have a mutation in katG, inhA, and/or rpoB genes or upon special request. Mutations gyrA and gyrB are known to be associated with resistance to fluoroquinolones.

Growth detection: Primary specimens are decontaminated, digested and concentrated daily. Each specimen is inoculated into a BD Mycobacterial Growth Indicator Tube (MGIT™) and a Middlebrook 7H10 agar plate for growth detection. Negative cultures are reported after 6 weeks of incubation.

When growth is detected, real-time PCR is performed to detect the presence of M. tuberculosis complex (MTBC) and/or M. avium complex. If positive for MTBC, identification of MTB complex member and antimicrobial susceptibility testing is completed.

Mycobacterial Isolate Identification

The Wadsworth Center Mycobacteriology Laboratory receives mycobacterial isolates for identification and susceptibility testing from clinical laboratories throughout New York State and out of state. Identification is performed using multiple methods, including real-time PCR, mass spectrometry and DNA sequencing (if needed).

Within 48 hours of receipt of an isolate, real-time PCR is completed to rule out Mycobacterium tuberculosis complex(MTBC) and M. avium complex (MAC). If positive for MTBC, further PCR testing is performed to identify the species within the TB complex.

For isolates that are negative for MTBC and MAC, real-time PCR is completed to rule out Mycobacteriumabscessus.

Isolates negative for M. abscessus are tested for identification using MALDI-TOF mass spectrometry.

Isolates not identified by MALDI-TOF are further tested using gene sequence analysis of rpoB, hsp65, and 16S rRNA for identification

Mycobacterium tuberculosis complex drug susceptibility testing

First line drugs: M. tuberculosis complex organisms are tested for susceptibility to the first-line TB drugs streptomycin, isoniazid, rifampin, ethambutol and pyrazinamide using BACTEC™ MGIT™ 960 Mycobacterial Detection System, an automated culture-based method. This method requires adequate growth of M. tuberculosis complex before testing can begin. Once growth is observed, the first-line susceptibility results are usually complete in approximately 2 weeks.

Second-line drugs: If resistance to the first line drugs is detected, susceptibility testing for 2nd-line drugs using agar proportion method is performed. The panel includes the antibiotics capreomycin, cycloserine, ethionamide, kanamycin, para-aminosalicylic acid, amikacin, rifabutin and ofloxacin. Agar proportion testing requires at least 3 weeks before final results can be issued.

Molecular susceptibility testing: Pyrosequence analysis of a portion of the katG, inhA, gyrA, gyrB and rpoB genes is performed on all MTBC isolates exhibiting resistance to first line TB drug(s) by conventional testing. This assay can also be performed on other isolates upon special request.

Sanger DNA sequencing of the pncA gene is performed on all patient specimens that exhibit PZA resistance.

Mycobacterium abscessus susceptibility testing

The Wadsworth Center Mycobacteriology Laboratory offers drug susceptibility testing for Mycobacterium abscessus since this organism is often resistant to common antibiotics. Susceptibility testing is done utilizing both real-time PCR to detect the erm 41 gene variants and broth microdilution susceptibility testing for the following drugs: clarithromycin, linezolid, moxifloxacin, cefoxitin, and amikacin.